Introduction to Biological and Small Molecule Drug Research and Development: Theory and Case Studies

Fame.

Preface

The history of medicines has been documented in detail but even at a broad and superficial level, different eras emerge quite clearly. First of all, for almost 2000 years, European medicine was dominated by Galen’s theory of the humours. This held that health depended on the correct balance of the four ‘humours’, or ‘principal fluids’ namely black bile, yellow bile, phlegm and blood produced in the body. The correct balance could be maintained, it was thought, by diet, blood-letting and medicines. The medicines were natural products used as infusions and mixtures based primarily on folklore; the early applications of morphine and quinine quickly come to mind. Indeed, some traditional therapies are still very popular and the practice has become part of the field of ‘complementary medicine’.

However, as it became possible to define detailed mechanisms of infection and the causes of disease, it was then feasible to design biological assays which could lead scientists to new therapies. Still, natural products provided a good proportion of the new medicines (for example, penicillins and steroids) although, increasingly, these natural materials were modified to provide optimized drug substances. In the latter half of the twentieth century, the naturally occurring small-molecule neurotransmitters were being modified to provide blockbuster drugs in areas such as heart disease, asthma and the treatment of stomach ulcers.

Medicinal chemistry came to the forefront at this time; chemists manufactured molecules on a small scale to test in the increasingly rapid (fast-throughput) assays. Novel, small molecules were preferred to allow patent protection and easy production at the multi-kilogram/tonne scale, respectively. Those entities passing the stringent safety requirements emerged as the drugs for the clinic. Still, today, small-molecule chemotherapeutics are sought after to provide greater protection against cancer and heart disease inter alia as well as debilitating conditions such as arthritis and dementia.

The contemporaneous development of protein chemistry allowed for the identification, isolation, purification and use of large biomolecules as drugs, for example, porcine insulin. With the advent of genetic engineering, the commercial production of biomolecules ex vivo became possible, for example, the production of human insulin in the bacterium Escherichia coli and erythropoietin in mammalian cells. A further seminal advance was the introduction of hybridoma technology that allows for the generation of human monoclonal antibodies of predefined specificity on an industrial scale.

This overall ability more efficiently to prepare selected proteins has given rise, particularly between 1991 and 2010, to a complementary set of medicines, commonly known as biopharmaceutics or biopharmaceuticals. Currently only ca 15% of medicines come into this bracket, but the way things are progressing, specifically the number of biopharmaceuticals presently in clinical trials and the proportion of biopharmaceuticals in the market place, this percentage could double by 2025.

It has been a fascinating challenge for us to present the background to these two types of manufactured medicine for a reader who is becoming interested in medicinal science. We start with a review of biological targets and the strategies to search for small molecules that might be therapies for a particular disease. Having established that base, the structures and properties of biopharmaceuticals are described before the two approaches are compared from scientific and commercial perspectives.

The case studies that are provided in the later chapters serve to illustrate the general concepts with specific examples. Where possible, large and small molecules targeting related therapies are grouped together. Overall, the coverage encompasses major therapeutic sectors as well as niche and orphan diseases. The clinical experience with the new drugs is outlined in some detail in most cases.

The Editors are extremely fortunate that the many world-leading experts agreed to contribute to this work. We are most grateful for their expositions, particularly in the chapters which describe complex issues, wherein great effort has been made to employ comprehensible language, keeping jargon to a minimum.

We hope that the book will provide a good basis to allow the reader to start to compare and contrast the science of small-molecule chemotherapies with that of the peptide/protein biopharmaceuticals.

C. Robin Ganellin

Roy Jefferis

Stanley M. Roberts

Chapter 1

Introduction to enzymes, receptors and the action of small molecule drugs

Stanley M. Roberts∗ and Alasdair J. Gibb†,    ∗School of Chemistry, Manchester University, Manchester M1 7ND, UK,    †Research Department of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6BT, UK

Abstract

Increasingly, pharmaceutical research and development is based on a detailed understanding of molecular interactions in diseased and healthy states of the human body. Over the past 50 years, most drug research has concentrated on the effects of small molecules on naturally occurring entities called enzymes and receptors. Hence, this chapter commences with an overview of the interactions of low-molecular-weight compounds (some natural [e.g. neurotransmitters] and some non-natural [e.g. drugs that inhibit certain enzymes]) with these natural macromolecules. This high-level introduction is followed by a more detailed inspection of the structures of some typical enzymes and receptors, emphasizing the complex shapes and subtle intermolecular interactions of these high-molecular-weight proteins. In addition, the importance of understanding the ‘on–off’ interaction between a small molecule and the target protein is illustrated by introducing the rate equations which dictate the kinetics of these episodes. The concluding section provides the first insight into the problems that have to be faced and overcome in moving from the point of having a compound with the desired effect on an enzyme or receptor in vitro to the position of introducing a useful drug to the marketplace.

Keywords/Abbreviations

Nerve cell; Neurotransmitter; Acetylcholine (receptor) (ACh(R)); Noradrenaline (NorA); Serotonin/5-hydroxytryptamine (5-HT); Dopamine (DA); Agonist/antagonist; Cyclic adenosine-3′,5′-monophosphate (cyclic-AMP); Cyclic guanosine -3′,5′-monophosphate (cyclic GMP); Adenosine/guanosine triphosphate (A/GTP); Catechol O-methyl transferase (COMT); Selective serotonin reuptake inhibitor (SSRI); Nicotinic/muscarinic AChR; Prodrug; Enzyme denaturation; Enzyme active/binding/catalytic sites; Co-enzyme; Enzyme cofactor; Michaelis–Menten equation; Lineweaver-Burk plot; L-DOPA; Allosteric enzyme inhibitor; Guanosine diphosphate (GDP); Hill-Langmuir equation; Scratchard plot; Lysergic acid diethylamide (LSD); G-protein coupled receptor (GPCR); Transmembrane (TM) domain; Gamma-aminobutyric acid (GABA); N-methyl-D-aspartate (NMDA); Sino-atrial (SA) node cell; Schild equation/plot; Efficacy

Chapter Outline

1.1 Section I: Background Information

1.1.1 Communication between cells: the roles of receptors and enzymes

1.1.2 Neurotransmitters, receptors and the nervous system

1.1.3 Introduction to enzymes and enzyme inhibitors

1.1.4 Other types of bioactive molecules

1.1.5 Factors influencing drug action

1.1.6 The impact of the sequencing of the human genome

1.2 Section II: More About Enzymes

1.2.1 Configuration of enzymes

1.2.2 Enzyme specificity, classification and nomenclature

1.2.3 Characteristics of enzyme catalysis

1.2.4 Enzyme reaction rates

1.2.5 Enzyme substrates as drugs

1.2.6 Enzyme inhibition and enzyme inhibitors as drugs

1.2.6.1 Irreversible inhibitors

1.2.6.2 Competitive inhibitors

1.2.6.3 Noncompetitive inhibitors

1.2.7 Enzyme regulation

1.3 Section III: More About Receptors

1.3.1 Bioassay and the measurement of drug effects

1.3.1.1 Bioassay

1.3.2 Quantifying drug–receptor interactions

1.3.3 Radioligand-binding studies – a direct measure of occupancy

1.3.4 Receptor structure

1.3.4.1 Nicotinic AChR structure: a ligand-gated ion channel

1.3.4.2 β-adrenoceptor structure: a G-protein-coupled receptor

1.3.5 Relating occupancy to response

1.3.4.1 Ligand-gated ion channels

1.3.4.2 Receptor mechanisms that involve second messengers

1.3.6 Competitive antagonism and the Schild equation

1.3.6.1 Drug blockade of open ion channels: a noncompetitive antagonism

1.3.7 Desensitization and the control of receptor number

1.3.8 Partial agonists, agonist efficacy and inverse agonism

1.4 Section IV

1.4.1 Conclusions: uncertainties in drug design and development

Further Reading

Acknowledgement

The section ‘More About Enzymes’ was adapted from Chapter 2 of the book ‘Medicinal Chemistry: the Role of Organic Chemistry in Drug Research’ (Academic Press, 1992) written by Dr Michael G. Davis. We acknowledge Dr Davis’s contribution.

1.1 Section I: Background Information

This chapter is adapted from the first three chapters of the book ‘Medicinal Chemistry: the Role of Organic Chemistry in Drug Research’ (eds. C. R. Ganellin and S. M.Roberts) Academic Press, London, 1992. While the basic principles remain the same, the text has been updated and modified to reflect the different focus of this book.

1.1.1 Communication between cells: the roles of receptors and enzymes

Some forms of life are composed of a single independent cell (the protozoa), while mammals are multicellular organisms. In between these two extremes there are life forms of varying complexity. All these organisms possess cell(s) to compartmentalize various chemical reactions in order to use available materials for energy and the maintenance of life’s processes.

Cells of different life forms have different characteristics (Figure 1.1) and, indeed, different cells from the same organism can be distinguished readily. For example, mammalian cells come in all shapes and sizes: compare the spheroidal leucocyte (the white blood cell), the flat epithelial cells found lining the mouth and the nerve cell (Figure 1.2).

FIGURE 1.1 Simplistic representations of a prokaryotic bacterial cell (a) and a eukaryotic (possessing a nucleus) human cell (b) Not all substructures are shown.

FIGURE 1.2 Shapes and sizes of mammalian cells.

The cells are organized such that chemical transformations can be accomplished efficiently, the rate of these transformations being controlled by Nature’s catalysts – enzymes. Enzymes are high-molecular-weight compounds which catalyse anabolic (synthesis) and catabolic (degradation) reactions. The trivial name of the enzyme often gives a guide to its role (see Eqn 1.1–1.3); a more comprehensive list of enzyme activities is contained in Section 1.2.

(1.1)

(1.2)

(1.3)

In order to coordinate their activities, the different cells in multicellular organisms need to communicate and this correspondence is accomplished mainly by small chemical molecules. For example, on receiving the appropriate signal, nerve terminals may release substances such as acetylcholine (ACh) (1), noradrenaline¹ (NorA) (2), serotonin (3) (otherwise known as 5-hydroxytryptamine or 5-HT) or dopamine (DA) (4), and these substances, known as neurotransmitters, can interact with the appropriate receptors.

The receptors can lie, for example, on the surface of the cells opposite the nerve terminal (Figure 1.3). The interaction of a neurotransmitter (agonist²) with its receptor usually effects a change in conformation of the macromolecular receptor, leading to a change in enzyme activity within the cell (Figure 1.4), and/or movement of ions into or out of the cell (Figure 1.5).

FIGURE 1.3 A neuroeffector junction (synapse).

FIGURE 1.4 Activation of an enzyme by occupation of a receptor by an agonist. (a) Receptor free, enzyme inactive. (b) Receptor occupied, enzyme triggered into action (allosteric activation of enzyme). (c) Agonist leaves receptor surface and enzyme quickly returns to inactive form.

FIGURE 1.5 Opening of an ion channel by the occupation of a receptor by an agonist. (a) Receptor free, channel closed. (b) Receptor occupied, channel opened, ion migration rapidly takes place down the electrochemical gradient. (c) Channel closes, neurotransmitter diffuses away. Difference in metal ion (e.g. Na+) concentration across the membrane is re-established by exergonic metal–ion pump.

One specific example of the process diagrammatically illustrated in Figure 1.4 is given by NorA, which will act on a receptor (for detail see Section 1.3.4.2 describing the G-protein-coupled receptor (GPCR)) basically resulting in the activation of the intracellular enzyme adenylate cyclase to produce cyclic adenosine 3′,5′-monophosphate (6) (cyclic AMP) from adenosine triphosphate (ATP) (5) as shown in Eqn (1.4).

Cyclic AMP initiates a cascade of other enzyme activations leading to the observed biological response. Cyclic AMP is inactivated by a phosphodiesterase enzyme (Eqn (1.4)).

(1.4)

An example of the process represented in Figure 1.5 is the interaction of ACh with its receptor which causes opening of the receptor ion channel (i.e. it is a ligand-gated ion channel – for more details see Section 1.3.4.1) resulting in the movement of sodium ions into the cell. Figures 1.4 and 1.5 illustrate the general idea that receptor occupation may result in an effect (e.g. change in enzyme activity) that can last for the lifetime of the occupation or the effect (e.g. ion movement) may be triggered and will not re-occur (for example due to receptor ‘desensitization’ – see also Sections 1.3.2 and 1.3.7) until disengagement of the agonist, re-priming of the system and re-engagement of the agonist and the receptor.

Note that if a neurotransmitter remains in the synaptic cleft, disengagement/re-engagement will continue and the receptor will be activated repeatedly; this will carry on until the chemical diffuses away from the site. To allow a faster return to the resting state after the neurotransmitter has ceased to be released from the nerve terminal, an enzyme may be present which will convert the neurotransmitter into an inactive substance or the neurotransmitter or hormone may be removed from the extracellular space by active transport (‘uptake’) into nearby cells. For example, ACh is deactivated by an esterase (Eqn (1.5)), while NorA is rendered inactive by methylation of one of the phenolic groups (Eqn (1.6)) through the enzyme catechol O-methyltransferase.

(1.5)

(1.6)

Most neurotransmitters including 5-HT and NorA are removed by uptake into the presynaptic terminal glial cells; indeed, the majority of the NorA released from a nerve terminal is removed from the synapse by this method. Drugs have been developed to slow down the process of uptake of a particular neurotransmitter into the presynaptic terminal. Selective serotonin reuptake inhibitors such as fluoxetine (Prozac) (7) have been developed as treatments for depression.

These examples illustrate that enzymes are not always contained within cells. Equally, not all chemical messengers are released from nerve terminals to act on adjacent terminals before being degraded. For example, adrenaline (8), NorA (2) and various steroids are released into the circulation from endocrine glands. Local hormones or autocoids such as histamine (a key component in the inflammatory response) are released from cells and travel through extracellular fluid to act on nearby cells. The three types of intercellular communication processes are shown in Figure 1.6.

FIGURE 1.6 Intercellular communication processes. (a) Nerve releases a neurotransmitter substance, e.g. ACh, which diffuses across a synapse to act on a postsynaptic membrane. (b) Endocrine gland releases a hormone, e.g. steroid, which is distributed throughout the body by the circulatory system. (c) Local hormone (autocoid) is released by cells and diffuses through the extracellular space to act locally, e.g. histamine released from mast cell or cells in the stomach.

It is important to understand that enzymes and receptors are both composed of amino acids condensed into high-molecular-weight polypeptide chains and can be associated with ions and small molecules; however, the likeness ends here. One of the major differences is that enzymes catalyse bond-making and bond-breaking reactions, while the receptors release the agonist unchanged.

In the normal healthy state, all cells are communicating, synthesizing and degrading molecules and changing ion concentrations for the overall well-being of the organism. As a result of disease, damage or degeneration, cellular activities may become impaired and the correct dynamic equilibrium must then be re-instated by means of a suitable drug. (Sometimes it may also be desirable to alter the normal physiology (in anaesthesia for example) by administration of a drug substance.)

It may be desirable to amplify the effect of a neurotransmitter. This can be accomplished by the following:

1. Increasing the concentration of the natural neurotransmitter by (a) direct supplementation through introduction of the substance into the body, (b) inhibition of enzymes that degrade the transmitter; or (c) inhibition of reuptake.

2. Using a more-potent and/or less readily metabolized surrogate of the natural substance (an unnatural agonist).

Alternatively, it may be prudent to decrease the effect of a particular neurotransmitter or hormone at a given receptor. This can be done using an antagonist substance, i.e. an unnatural compound which will bind strongly to a receptor without eliciting a response and which will prevent access of the natural agonist to the receptor.

An antagonist diminishes or abolishes the effects of the corresponding agonist. Two types of antagonists are known: a competitive antagonist competes with the agonist for a binding site at the active site of the receptor (also known as the orthosteric site), while a noncompetitive antagonist binds at a different site (an allosteric site) from that of the agonist. In the former situation, the effect of the antagonist decreases in the presence of increasing concentrations of agonist, while in the latter situation, the effect of the antagonist is often independent of agonist concentration.

Similarly, certain enzyme substrates and specific or highly selective enzyme inhibitors can prove to be useful drug substances. The inhibition of human immunodeficiency virus (HIV) protease is one example (see Chapter 13), complementing drugs such as azidothymidine which block another HIV enzyme called reverse transcriptase.

Before amplification of these points, the central control of cell communication and a more detailed consideration of certain neurotransmitters and receptors are warranted.

1.1.2 Neurotransmitters, receptors and the nervous system

In complex organisms such as man, there are a considerable number of receptors and a multitude of different enzymes. Actions are coordinated by the central nervous system (CNS) (the brain and the spinal cord) and some actions are the result of sensory input (sight, sounds, touch, hearing, etc.). Output from the CNS is directed towards the autonomic nervous system (the sympathetic and parasympathetic systems) and nerves associated with voluntary motor functions as illustrated in Figure 1.7. Voluntary motor function deals with the controlled movement of muscles (skeletal muscle) and the associated limbs; some of the organs controlled by the parasympathetic and sympathetic nerves are listed in Figure 1.8 and the effects on selected organs from the two systems are listed in Table 1.1. In short, the sympathetic and parasympathetic systems operate in a complementary fashion. In response to a particular external influence, enhanced stimulation of the sympathetic nervous system occurs and leads to preparation for ‘fight or flight’ (Figure 1.9). In the relaxed state (Figure 1.10), stimulation of the parasympathetic nervous system predominates and deals with secretion and voidance of materials from the body.

FIGURE 1.7 The peripheral nervous system.

FIGURE 1.8 Organs controlled by sympathetic (S) and parasympathetic (P) nerves.

Table 1.1

Response to Activation of Sympathetic and Parasympathetic Nervous System

FIGURE 1.9 Stimulation of the sympathetic nervous system due to fright leads to preparation of the system for flight or fight increase in heart rate, dilation of bronchi, dilation of pupils, constriction of peripheral blood vessels (pallor), etc. Source: From B.L.A.T. Booklet ‘Action of Drugs’, Centre for Health and Medical Education, London.

FIGURE 1.10 In the relaxed state, stimulation of the parasympathetic nervous system is predominant and leads to (inter alia) slowing of the heart and increase in the activity of the gastrointestinal tract. Source: From B.L.A.T. Booklet ‘Action of Drugs’, Centre for Health and Medical Education, London.

Some important neurojunctions and the associated neurotransmitters in the nervous system are shown in Figure 1.11.

FIGURE 1.11 Neurotransmitters in the peripheral nervous system. Asterisk indicates release into the circulation to act on distant receptors in the periphery. S, sympathetic nervous system; P, parasympathetic nervous system; V, voluntary motor function; ACh, acetylcholine; NorA noradrenaline; Mus, muscarinic receptor; Nic, nicotinic receptor.

Note that the acetylcholine receptors (AChRs) are divided into two categories, the classification being based on the actions of two drugs of plant origin.³ The receptors which are activated by muscarine are termed muscarinic receptors and those activated by binding nicotine are called nicotinic receptors. Thus nicotine and muscarine mimic the action of ACh at two different distinct receptors. The effects on various end organs of stimulation of the appropriate AChRs are listed in Table 1.2. Inhibition of the action of ACh at the neuromuscular junction leads to muscle relaxation.

Table 1.2

Situation of Some Acetylcholine-Controlled Neuroeffector Junctions

NorA and adrenaline stimulate adrenoceptors. When NorA is released from a presynaptic nerve terminal, it crosses the synaptic cleft and initiates a response in the postsynaptic tissue by combining with one of two types of receptor called α-adrenoceptors and β-adrenoceptors. The type of receptor found postsynaptically to noradrenergic nerves in the sympathetic system depends on the type of tissue; classification of adrenoceptors in different tissues is again based on the ability of the agonists to initiate responses and antagonists to prevent responses. A further subclassification of β-adrenoceptors has been made: in man the majority of β-adrenoceptors in the heart are called β1-adrenoceptors and these are distinct from other β-adrenoceptors (dubbed β2-adrenoceptors) found elsewhere in the periphery (outside the CNS). Many tissues have a mixed population of β1- and β2-receptors. NorA and adrenaline have different effects on α- and β-receptors; NorA is potent at stimulating α-receptors but is less potent at activating β-receptors, while adrenaline elicits activity from both α-and β-receptors at about the same level.

Some important sites of α- and β-adrenergic receptors are given in Table 1.3.

Table 1.3

Important Adrenaline and Noradrenaline Receptors

Note that activation of α-receptors generally results in a stimulant response (except in the gut), while activation of β-receptors leads to an inhibitory response, namely, relaxation of muscle (except in the heart). Blocking of α- and β-receptors causes, inter alia, relaxation of peripheral blood vessels and slowing of the heart, respectively, and can have beneficial effects in the treatment of angina and hypertension, while β2-stimulants can alleviate mild to moderate asthmatic attacks by relaxation of bronchiolar muscle and widening of airways. Thus the antiasthma drug salbutamol (Ventolin) (9) is a β2 agonist while Propranolol (10) was one of the first ‘beta-blockers’ useful for the treatment of hypertension.

Some peptides act as neurotransmitters. For example, receptors for the enkephalins (11) have been demonstrated within the CNS. It is believed that morphine (12) and the other opiates exert their analgesic action by interaction with these receptors. Thus, morphine (12), heroin (13) and codeine (14) can be considered to be agonists at the enkephalin receptor; other compounds such as the painkilling drug buprenorphine are partial agonists, i.e. have mixed agonist and antagonist properties. Opiate receptor blockers (antagonists), e.g. naloxone, are also known.

Histamine (15) is a neurotransmitter which also occurs in many tissues in the body. It is stored in mast cells and platelets and is released from these sites in response to stimuli such as allergic reactions and injury. Four main types of histamine receptors, termed H1, H2, H3 and H4, have been identified; they differ in sensitivity to various unnatural agonists and antagonists. Bronchiolar smooth muscle has H1 receptors; activation by histamine causes contraction of the muscle and bronchoconstriction. The actions of histamine on vascular smooth muscle are complex, species dependent and mediated by both H1 and H2 receptors, while gastric acid secretion from parietal cells is stimulated by mucosally released histamine acting at H2 receptors. H2 receptor blockers, such as cimetidine and ranitidine are useful in the treatment of conditions in which there is excess acid secretion in the stomach, especially in duodenal ulceration. The H3 receptors appear to be autoreceptors which inhibit further histamine synthesis or release; they occur especially in nervous tissue, while H4 receptors are involved in immunologically based responses.

It is noteworthy that all the previously mentioned transmitters, i.e. ACh, NorA, 5-HT, enkephalins and dopamine, as well as histamine and adrenaline, have receptors in the CNS. Interaction with these receptors will elicit a response and if interaction of a potential drug substance with peripheral receptors (e.g. at the neuromuscular junction, on bronchiolar smooth muscle, in parietal cells, in heart muscle, on blood vessels etc.) is beneficial, then it is often necessary to ensure that the compound does not cross the ‘blood–brain barrier’ to cause unwanted side effects through interaction with receptors in the CNS. Of course, if a drug has its effect by interacting with receptors in the brain (e.g. an antidepressant), the compound must have the appropriate physical properties (high lipophilicity, low polarity) in order to cross the blood–brain barrier.

1.1.3 Introduction to enzymes and enzyme inhibitors

The active centres of enzymes are similar in many ways to the agonist-binding sites of receptors, and enzyme inhibitors are identical in principle to receptor blockers. Simple inhibitors derange the active centre by engaging it directly (isosteric inhibition) or by inducing a conformational change affecting the active site through binding to a distant site (allosteric inhibition) (see Section 1.2.7). Unnatural substrates for an enzyme which are slowly processed are also effective inhibitors of the physiological enzyme action provided that they have a substantially greater affinity than the natural substrate for the enzyme centre. Substantial inhibition of a key enzyme-controlled process in an organism will generally lead to the demise of the organism. If the enzyme in question is peculiar to a bacterium or fungus that has invaded the mammalian host, then inhibition will eradicate the pathogen and leave the host unharmed. For example, penicillins and cephalosporins act as highly selective antibacterials because they inhibit an enzyme that is found only in the cell wall of bacteria.

1.1.4 Other types of bioactive molecules

Not all drugs act on discrete receptors or at active sites of particular enzymes. Others, such as some general anaesthetics, act partly by insertion in lipid membranes, thereby modifying ion transport across the membrane. Yet others, such as antacids or some diuretics, produce their effects by means of their physicochemical properties. Many steroid drugs and hormones must first pass into cells, becoming associated with specific cytosolic proteins which facilitate their transport into the cell nucleus where gene expression, and ultimately protein synthesis, is modified. Facilitated transport is also crucial in getting polar building blocks (e.g. nucleosides) across the hydrophobic blood–brain